Structural Basis of X-ray-Induced Transient Photobleaching in a Photoactivatable Green Fluorescent Protein
Structural Basis of X-ray-Induced Transient Photobleaching in a Photoactivatable Green Fluorescent Protein
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DOI：
10.1021/ja907296v
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发表时间：
2009-12-23
影响因子：
15
通讯作者：
Bourgeois, Dominique
中科院分区：
文献类型：
--
作者：
Adam, Virgile;Carpentier, Philippe;Bourgeois, Dominique
我们观察到光激活荧光蛋白 IrisFP 处于瞬态黑暗状态，具有接近原子分辨率。这种暗态被分配给一种自由基物种，该自由基物种要么弛豫到基态，要么演变成永久漂白的发色团。我们利用 X 射线填充自由基，该自由基可能是在可见光照射下通过三重态电子转移反应形成的。组合的 X 射线衍射和晶体紫外可见吸收、荧光和拉曼数据表明，IrisFP 中的自由基形成涉及显着但可逆的发色团变形，表明 17 共轭的瞬时损失。这些结果表明，发色团的亚甲基桥是荧光蛋白的致命弱点，有助于揭示 FP 中的眨眼和光漂白机制，这对于光稳定性变体的合理设计具有重要意义。
We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystalto UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of 17 conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants.